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Understanding the molecular pathways coordinating protein biosynthesis and trafficking by aphids and the involvement of endosymbiont bacterium, Buchnera aphidicola in those processes, is critical to discovering the mechanisms of virus transmission by aphids. Ultimately this knowledge can be used to develop targeted approaches for disrupting the spread of insect-vectored viruses. Yellow dwarf viruses are vectored by the green bug aphid, Schizaphis graminum. Yellow dwarf infections devastate cereal crops by greatly reducing seed yield. Here, we report the application of a novel reverse phase (RP) separation using the different ion pairing agents, ammonium acetate or triethylammonium acetate and the organic modifier methanol as the first dimension separation followed by an online, low pH RP separation using formic acid and acetonitrile, employing data independent mass analysis, MSe, for protein identification. Proteomics studies in the green bug are challenging due to the lack of genomic resources. However, the genome of a closely related species, Acrythosiphon pisum is available and facilitated protein identification by homology-based searching. Nearly 1000 proteins were identified including 190 hypothetical proteins and 80 proteins from the endosymbiont Buchnera. Several proteins previously associated with virus transmission were identified, such as GroEL, actin, cuticle proteins, HSP70, GAPDH3, and even the low abundance proteins RACK-1, and cyclophilin, demonstrating the utility of this workflow for the discovery of proteins involved in virus transmission by aphids. The new 2-dimentional RP-RP (RP2) separation combined with the enhanced sampling and detection of peptides provided by MSe enabled us to assign specific protein functions to 20% of the proteins identified, most of which were from the green bug proteome. Furthermore, the RP2 technique effectively dealt with the wide dynamic range of the sample, as we detected approximately 50 low abundance proteins from a pathogenic fungus of insects, previously uncharacterized in our green bug population.